Advanced Oligonucleotide Characterization Using Multi-Reflecting Time-of-Flight Technology

Importance of the topic

Oligonucleotide therapeutics rely on accurate characterization to ensure safety, stability, and efficacy throughout development and commercialization. High-resolution LC-MS methods are essential to confirm full-length product identity, detect synthesis-related impurities, and monitor degradation by-products. Reliable analytical workflows reduce the risk of undetected variants that could compromise therapeutic performance and regulatory compliance.

Study objectives and overview

This application note evaluates the performance of the ACQUITY Premier UPLC System coupled to the Xevo MRT Mass Spectrometer for detailed analysis of a phosphorothioate antisense oligonucleotide (GEM 91). By leveraging waters_connect software with INTACT Mass and CONFIRM Sequence applications, the study aims to demonstrate robust intact mass confirmation, impurity profiling, and sequence verification using data-independent fragmentation (MSE).

Methodology and instrumentation

An ion-pair reversed-phase LC-MS method was employed in negative electrospray ionization mode. Mobile phases consisted of triethylamine (TEA) and IonHance HFIP in water or methanol. Chromatographic separation used an ACQUITY Premier Oligonucleotide C18 column at 60 °C. Sample introduction utilized the ACQUITY Premier UPLC System with a 5 µL injection and a 0.4 mL/min flow rate. Mass analysis was performed on the Xevo MRT MS, featuring multi-reflecting TOF geometry. The system operated at 10 Hz scan rate across 400–4000 m/z, achieving up to 100 000 FWHM resolution and sub-ppm mass accuracy. Data processing integrated waters_connect INTACT Mass App for automated peak detection and deconvolution, and CONFIRM Sequence App for data-independent fragmentation analysis.

Main results and discussion

The workflow achieved consistent mass accuracy below 2 ppm for the full-length GEM 91 product and identified nine impurities down to 0.1 % abundance. High-resolution spectra (approximately 100 000 FWHM) resolved coeluting impurities differing by only 0.05 m/z. The INTACT Mass App automated deconvolution confirmed the oligonucleotide mass and purity profile, while the CONFIRM Sequence App delivered 100 % sequence coverage with fragment mass errors under 2 ppm. Efficient desolvation and minimal solvent adducts ensured clear isotopic resolution and low depurination artifacts.

Benefits and practical applications

• Sub-ppm mass accuracy and high resolution enable detection of subtle modifications and low–abundance impurities.
• Automated workflows reduce manual data review and transcription errors, increasing throughput.
• Data-independent fragmentation processing ensures comprehensive sequence verification without precursor selection bias.
• Compliance-ready, app-based informatics streamlines acquisition, processing, and reporting for regulated environments.

Future trends and potential uses

The integration of multi-reflecting TOF technology with advanced informatics paves the way for real-time monitoring of oligonucleotide production. Future developments may include higher multiplexing capabilities, coupling with orthogonal separation techniques, and incorporating machine learning for automated interpretation of complex fragmentation patterns. Such advances will support the growing demand for rapid, high-confidence quality control in oligonucleotide therapeutics.

Conclusion

The combination of the ACQUITY Premier UPLC System, Xevo MRT MS, and waters_connect software delivers a robust, sensitive, and accurate platform for oligonucleotide characterization. This streamlined approach enhances confidence in full-length product confirmation, impurity profiling, and sequence verification, facilitating faster decision-making in therapeutic development and ensuring high-quality outcomes.

References

1. Waters Corporation. Intact Mass Confirmation Analysis on the BioAccord LC-MS System for a Variety of Extensively Modified Oligonucleotides. Application Note, 2020.
2. Waters Corporation. A Xevo G3-based Workflow for Purity Determination, Intact Mass Measurement, and MS/MS Sequencing of Impurities Detected in Synthetic Oligonucleotides. Application Note, 2024.
3. Waters Corporation. An automated, compliance-ready LC-MS workflow for intact mass confirmation and purity analysis of oligonucleotides. Application Note, 2020.